This invention relates to microscopy and more particularly to the calibration of fluorescence detection microscopes, to the calibration of on-axis, wide field of view scanning fluorescence microscopes, and ultimately to quantified fluorescent microscopy having application from the forefront of genomic research and drug discovery to clinical use. The invention has particular application to the accurate reading of biochips and micro arrays.
Testing or calibration targets are employed to evaluate system performance of conventional microscopes. These are used to establish a baseline between different microscope systems and to characterize image quality in terms of its conventional components: resolution, contrast, depth of field and distortion. Common offerings for conventional microscopy are the USAF Field Resolution target, the USAF Contrast Resolution Target, the Star Target, the Ronchi Ruling Targets, Modulation Transfer Function Targets, Depth of Field Targets, and Distortion and Aberration Targets. There are others.
The targets are typically printed or vapor deposited patterns on plastic or glass substrates. The optical features on the target are preferably finer than the resolution of the optical system being tested. While it is desirable that the reference features have dimensions or parameters an order of magnitude smaller than those of the specimens to be examined by the microscope, practitioners have had to accept reference dimensions or parameters only 4 or 5 times smaller than those of the specimens to be examined.
Fluorescent microscopy of specimens is different from and more demanding than conventional microscopy because it is based on relatively low-level fluorescent emissions excited by illumination of the specimen, typically employing confocal arrangements for detecting the relatively weak signal through a pin hole or the like. An example is the detection of fluorescence from dried liquid spots containing possibly fluorescing biological material, the dried spots being essentially at the focal plane of the instrument (dried spot thickness less than a few microns). Another example is fluorescence from a biological microarray such as from a Microchip(copyright) biological array product, as produced by Affymetrix, Inc., in which the fluorescing material is of insignificant thickness.
For testing or calibration targets for fluorescence microscopy, besides the numerous conventional components of image quality, there is the requirement of testing the optical efficiency of the system in respect of fluorescence emission. This introduces significant complications, as fluorescence involves an excited photochemical effect, to produce a voltage or signal level in the detector, that introduces signal to noise ratio considerations that interact with measurements of the various optical components involved in the calibration. In general the signal to noise ratio must be at least 3 to 1 to obtain satisfactory operation.
It has been an unsolved problem, to find a calibration target that adequately simulates the fluorescent activity which it is desired to quantify over a broad range of instruments and conditions of use. It is wished to simulate fluorescing specimens that generally lie within the depth of field of the microscope, and in the case of micro dots of biological material, lie essentially at a plane, e.g. in a depth of only a few microns or even substantially less. As the dimensions of individual specimens to be imaged become increasingly smaller as microarray technology advances, the significance of not having a suitable calibration tool has become increasingly severe.
The difficulties for fluorescent microscopy is that, without the desired degree of calibration, it becomes difficult to compare the results obtained in biological or other research performed with different instruments, thus creating serious difficulties in comparing and coordinating the results of different laboratories, whether the laboratories be at different institutions, or separate laboratory facilities within the same institution. Likewise, even with a given instrument, the uncertainties of calibration can introduce errors in the measurement of important actions such as proportional expression, etc. In particular the lack of good reference and calibration is felt at the forefront of research where results are so new and there has been insufficient time or experience to generate reliable standards. The development of true quantified fluorescence microscopy can fulfill this need.
On the other hand, the availability of a strong calibration tool is seen to open the possibilities of inexpensive and reliable fluoresence instrumentation and procedures for the clinical setting for diagnosis and treatment.
Existing calibration tools for conventional microscopy do not satisfactorily fill the needs of fluorescence microscopy. A number of special techniques have been offered.
One, offered by Max Levy Reprographics, uses a layer of organic fluorescent material e.g. of 3 micron thickness, having fluorescence emission across a broad wavelength spectrum, deposited on a non-fluorescent glass substrate such as synthetic quartz. A suitable pattern is then etched away into the fluorescent material, so that the critical edges of the reference are defined by the exposed edges of the fluorescing material. I realize that one shortcoming of this technology is that the minimum thickness of the fluorescent material that can be deposited is of the order of 3 micron and, with such thickness, the edges of the pattern do not etch squarely. The finest reference details that can be formed in this material are believed to be approximately 4 micron width lines, spaced apart 8 microns on center. This is unsatisfactory for calibration with respect to instruments employing conventional 5 micron spot size and is an order of magnitude greater than required to evaluate optical spots that are xc2xd micron in diameter, achievable with a microscope having an 0.7 NA objective in air, or xc2xc micron diameter achievable with a 1.4 NA, oil immersion objective. The relatively large thickness of the fluorescent layer poses problems of edge definition, particularly because the fluorescent rays emit at acute angles to the surface and can be blocked by the edges of the material, or on the other hand, the edges themselves fluoresce, to produce confusion.
Another technique for testing a fluorescent microscope uses as a substrate a fluorescent glass on which is deposited a very thin metal layer e.g. a few hundred Angstrom thick. Preferably a nickel layer is employed. A suitable pattern is subsequently etched in the metal to create fine features, as small as xc2xd micron dimension. Whereas this technique does not have the foregoing edge problem, I have realized that there are shortcomings to this approach, owing to the fact that the glass constitutes a significant fluorescing volume, i.e., a substantial thickness, 1 millimeter, far exceeding the depth of field1, and the fluorescent radiation emitted from this volume causes focus to be difficult to define accurately and hence is an unsatisfactory standard for many purposes. Also, no presently known glass has a uniform, broad fluorescence spectrum.
1 For a spot size of 5 or 1xc2xd micron, the depth of field is typically about 50 micron and 4.5 micron, respectively, and progressively less for smaller spots toward which the field is trending. 
Yet another design, offered by Affymetrix, Inc., employs a fine-featured pattern etched in a thin metal layer, as described in the above paragraph, but the substrate is non-fluorescent quartz. The fluorescent emission is obtained from a fluid volume that bathes the pattern. I realize that this design suffers from the same shortcoming of employing a volume of fluorescing method as previously described, however, it does permit the ready use of a variety of fluorescent fluids, so that the reference may be matched to the particular study in question. Though a useful product, it comes short of offering a reliable fluorescent emission standard.
According to one aspect of the invention a calibration tool for fluorescent microscopy is provided comprising a support on which is carried a solid surface layer comprised of effective fluorophores, and a thin mask of non-fluorescent material defining reference feature openings of limited dimensions exposing portions of the fluorophore-comprising surface layer.
Preferred embodiments have one or more of the following features:
The support is flat and rigid.
The surface layer is opaque.
The mask comprises an etched thin metal film which is intimately engaged face-to-face with the solid surface layer that comprises effective fluorophores.
The tool has a transparent non-fluorescing transparent layer overlying the mask and the solid fluorophore surface layer, arranged so that the fluorophores are excited by radiation passing through the transparent, non-fluorescing layer.
The fluorophore-containing surface layer is directly exposed to exciting radiation through openings in the mask.
The solid surface layer is a broad band fluorescence emitter.
Either the thin metal film or the solid surface layer that comprises fluorophores is directly deposited upon the other.
The calibration tool is combined with a confocal microscope having a restricted depth of field and the solid surface layer that comprises fluorophores has an effective depth of less than the depth of field of the confocal microscope, preferably the surface layer having an effective fluorescent emittance that can produce a full scale response of the microscope.
The support is flat and rigid.
The solid surface layer is comprised of fluorescent polyimide.
The solid surface layer is comprised of a thin layer of fluorescent glass or glass-like material.
The solid surface layer is a congealed sol-gel coated layer, with fluorophores disposed in the coating layer.
The fluorophores comprise a dye, such as Cy3, Cy5 or fluorescene, which are fluorescent at a desired wavelength.
According to another aspect of the invention, a process for producing a calibration tool comprises providing in face-to-face contact, a support, an etched metal layer defining alignment features for fluorescent microscopy and a surface layer comprised of effective fluorophores.
Preferred embodiments have one or more of the following features:
The support is flat and rigid.
A uniform metal film is deposited on the face of the surface layer and subsequently etched to produce the pattern.
A surface layer containing effective fluorophores is deposited over a pattern-defining mask.
The surface layer is comprised of polyimide that fluoresces in response to excitation over a wide band of wavelengths.
According to another aspect of the invention, a method of quantified fluorescence microscopy is provided comprising providing a fluorescence detecting microscope, employing a calibration tool as described above to calibrate the microscope, and performing fluorescence microscopy of specimens employing the calibrated microscope. Preferably the microscope is an on-axis flying objective microscope, and most preferably, the microscope has a micro objective lens carried upon a rapidly oscillating rotary arm.
In a preferred embodiment, a fluorescent calibration tool is built with a suitably fluorescent solid surface layer of constant thickness that is opaque, made of organic material or inorganic material, carried on a suitable support. For materials that are not naturally opaque, dyes or pigments are added. A very thin metal layer is subsequently deposited on the opaque fluorescent material and covered with a layer of photo-resist. An appropriate pattern is then imaged on the photo-resist and chemically etched. The resulting fluorescent pattern showing through the etched openings has extremely fine features because the metal layer is as little as a fraction of a micron thick, preferably about 100 to 300 Angstrom thick.
The pattern-creating process can be identical to the process used to create integrated circuits. Presently that technology enables the formation of features as narrow as 0.2 micron width lines separated by spaces of the same dimension.
In the calibration tool of the present invention the fluorescence is caused to be a surface emission phenomenon, which permits reliable focusing and fluorescence calibration, that can be used as a standard, and enable all instruments to be set to the same standard.
Another advantage of the invention, is that very stable fluorescing material can be used, that is insensitive to photobleaching.
An important fluorescing material according to the invention, which has a broad band of fluorescent response, is a selected polyimide. The presently preferred choice is in the form of Kapton(trademark) available in liquid form and used for spin coating substrates and creating sheets with 1 to 10 micron thickness. A suitable product is available under the trade designation WE-IIII or PI-IIII from H. D. MicroSystems, Wilmington, Del. This material is a polyimide which has as a backbone a high molecular weight polyimic acid precursor comprised of specific aromatic decanydride and an aromatic diamine.
Another polyimide product, Probonide 116A, available from Arch Chemicals of Portsmouth, N.H., exhibits broad band fluorescence of approximately xc2xc the intensity of the H. D. MicroSystems product, that can be satisfactorily used. Its chemized structure is presented in its literature as: 
In certain cases the fluorescing polyimide material is one that is provided to the semiconductor industry as a self-priming, non-photosensitive polyimic acid formulation which becomes a fully stable polyimide coating after thermal curing.
Another material for the surface layer, suitable for a specific wavelength of interest, is an extremely thin layer of fluorescent glass deposited e.g. by evaporation or by a sol-gel process on a non-fluorescent support. In the case of a sol gel, large molecules of a glassy type of material are suspended, with selected fluorophores in a water or alcohol carrier, and applied as a film coating to a support. It is baked at a relatively low temperature to form a thin glassy fluorescent film. In these and other cases, fluorescing dyes for specific wavelengths are incorporated in a suitable non-fluorescing and preferably opaque binder, applied as a thin, uniform thickness coating. Examples of fluorescing dyes are Cy3, Cy5, and fluorescene.
In all events, the substance of the surface layer must be selected to produce sufficient fluorescence to be detected in the way that is normal to use in operating the instruments for examining fluorescent specimens. The specific selection of a fluorescing reference material is dependent upon numerous parameters such as the response of the instrument, the selected wavelength, the size of the features to be examined and the spot size of the excitation beam. In the case of the commercial instrument as described as an example in the accompanying appendix, the fluorescent material must produce of the order of one million times the radiation detected at the detector. By following such an analysis as provided in the Appendix, one may select an appropriate solid fluorescing material for the instrument and task at hand.
It is believed, however, that the polyimide materials described earlier above provide a great benefit over others in being broad band and hence suitable as a single reference that is useful over a range of selected wavelengths at which important experiments are performed.
Some fluorescent microscopy applications demand that the material under inspection be located behind a transparent protective window, typically made of non-fluorescent optical glass such as synthetic quartz. In such cases the alignment tool preferably duplicates the application and the metalized target is first created on the glass and the fluorescent media is applied as a coating covering the metalization as well as the glass, or is provided as a planar coating on a second optically flat member which is then mounted face-to-face with the metal layer on the first optically flat member.
It is an important aspect of the invention that the effective fluorophores2 for producing photons that reach the detector lie substantially only in a surface layer, thus approximating what occurs when dots of biological specimen material only a fraction of a micron thick produce fluorescence in response to an incident excitation beam. According to the invention, the limitation of fluorescence to the surface layer suitable for a given application is accomplished by one or a combination of a number of techniques. In one case, the binder material for forming the solid matrix in which the calibration fluorophores are contained, is made essentially opaque at the excitation or detection wavelength or both, such that a large fraction, e.g. 80% or more, preferably as much as 99% of the detected fluorescing radiation, emanates from a surface layer of depth, xcex94t, that is only of the order of the thickness of the specimen to be inspected, and within the depth of field of the instrument. In another case, the micro thickness of a layer in which the fluorophores are confined is controlled to a high degree of uniformity, the layer sitting on an opaque support devoid of fluorophores, such that even if some fluorescence occurs from a depth beyond the preferred bound, the resulting fraction of luminescence outside of the bound is uniform across the tool because of the uniformity of coating thickness, and hence is not effective to significantly disturb the calibration. In another case, the fluorophores are introduced to a surface layer after preforming the surface layer, e.g., by diffusion, spray or implantation techniques that confine the fluorophores essentially to the surface that is to be exposed by openings in the pattern.
2 As used in this specification, the term, xe2x80x9ceffective fluorophoresxe2x80x9d is meant to include substantially all of those fluorophores which are effective to produce meaningful fluorescent radiation from the face of the surface layer that can reach the detector of a microscope, and does not refer to fluorophores which are either out of the range of excitation radiation of the microscope due to the opacity of the matrix, or, though within the range of effective excitation radiation, do not produce fluorescent radiation that reaches the detector of the microscope, due, e.g., to absorption by the opaque matrix material. 
Thus, according to the invention, an effective solid fluorescent surface layer is provided that can serve as a proxy for the specimen to be examined.
The thickness of the thin metal layer or other material forming the reference pattern also matters, because many rays of the detected fluorescence form an angle as great as 45 degrees with the surface being examined, and can be blocked by the edge walls of the pattern elements, if the elements are too thick, to impair the resolution of detection of the pattern edge. The finer the features to be inspected, the finer must be the calibration of the instrument, hence the more critical becomes the thickness of the pattern elements. By use of a pattern produced by photolithographic or similar etching3 techniques it is possible to form a pattern of material of only a fraction of a micron thickness, the metal film being formed e.g., by chemical vapor deposition or sputter coating, which is then etched by photolithographic techniques to form the pattern of reference lines, circles, etc.
3 By xe2x80x9cetchingxe2x80x9d is meant a process capable of precisely removing material and includes for instance acid and other chemical etch, laser etching and etching by bombardment of energetic particles such as accelerated electrons or ions and may employ masks of high precision produced by optical, X-ray or ion beam lithography. 
Finally, also of concern is the wavelength of fluorescence produced by the fluorescing surface layer. According to one aspect of the invention, the calibration tool is made employing a broad band fluorescent material and thus is useful with various lasers and wavelengths used in a microscopes and with different types of fluorophores used in various lines of scientific or industrial inquiry. In one example a polyimide material is selected which has effective fluorescence for use as a reference at wavelengths from 473 Nanometer to 650 Nanometer, or more preferably from 450 Nanometer to 800 Nanometer, covering essentially the entire visible spectrum. (The visible spectrum is important, since a great deal of historical biological data has been generated in that region, and is available for reference and comparison as research proceeds.) However, it will be understood that certain broad aspects of the invention are not so limited, as fluorophores in the near infrared and ultraviolet may be employed, given suitable circumstances with respect to the biology and the available sources of illumination and detection.
Another aspect of the invention is the use of the calibration tool described in combination with a flying objective, on-axis scanner, to achieve highly reproducible quantified fluorescence microscopy. While microscopes with any means of moving the lens preferably a micro lens, is included, significant further advantages are obtainable by employing on oscillating rotary arm to transport the micro lens over the specimen or calibration tool.